Eptifibatide, also known as N6-(aminoiminomethyl)-N2-(3-mercapto-1-oxopropyl-L-lysylglycyl-L-α-aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic (1-6) disulfide, is a highly specific cyclic heptapeptide antagonist of the platelet glycoprotein IIb/IIIa used for the treatment of cardio vascular disease, is represented by the following Formula I.

Eptifibatide is a short acting parenteral antithrombotic agent that is used for treating Acute Coronary Syndrome (ACS). It is also used in patients undergoing Percutaneous Coronary Intervention (PCI). Eptifibatide is a platelet aggregation inhibitor (PAI) and belongs to a new class of RGD mimetics-arginine (R), glycine (G), aspartic acid (D). Eptifibatide reversibly inhibits platelet aggregation by preventing the binding of fibrinogen, von Willebrand factor and other adhesive ligands to the GP IIb/IIIa receptors.
Eptifibatide is marketed in the United States under the brand name of INTEGRILIN®, and is used to treat patients with acute coronary syndrome (unstable angina and non-Q-wave MI), including patients who are to be managed medically and those undergoing PCI. Integrilin is intended for use with acetylsalicyclic acid and unfractionated heparin.
In terms of peptide synthesis methodology, two major synthetic techniques dominate current practice. These are synthesis in solution phase and synthesis on solid phase. The solution phase synthesis has generally been viewed as more feasible than solid phase synthesis for the large scale manufacture of eptifibatide.
Alternatively, selected polypeptides are produced by expression of recombinant DNA constructs. The DNA encoding the sequenced polypeptide is prepared using commercially available nucleic acid synthetic method. Production by recombinant method is particularly preferred for peptides of at least 8 amino acid residues. U.S. Pat. Nos. 5,318,899 and 5,958,732 discloses recombinant techniques to synthesize eptifibatide; the lysine residue is converted to homoarginine residue by solution phase synthesis. WO 2005/121164 and US 2007/249806, disclose the synthesis of peptide on a solid support resin, and subsequently modifying by solution phase synthesis for conversion of lysine residue to homoarginine residue, through guanylation with 3,5-dimethylpyrazole-1-carboxamide nitrite.
In solid phase peptide synthesis, the desired peptide is prepared by the step-wise addition of amino acid moieties to a building peptide chain. The two most widely used protocols, in solid-phase synthesis, employ tert-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) as amino protecting groups. Many of the reported synthetic approaches to eptifibatide have employed known techniques of solid phase peptide synthesis, mainly, using Boc-chemistry, as described, for example, in U.S. Pat. No. 5,318,899, U.S. Pat. No. 5,686,570, U.S. Pat. No. 5,747,447, U.S. Pat. No. 5,759,999, U.S. Pat. No. 576,333, U.S. Pat. No. 5,770,564, U.S. Pat. No. 5,807,825 and U.S. Pat. No. 5,851,839. Fmoc based solid phase synthesis are described in WO2005/121164, WO2003/093302, CN1858060A, CN1500805A, WO2006045483, WO2006119388, WO2003093302 and US 2007249806. For the synthesis of cyclic peptide, the linear peptide conveniently prepared on resin is cleaved from the support followed by cyclization in solution.
However, as with many peptides the major challenge with eptifibatide is to produce sufficiently pure material at an acceptable cost. A commercial scale solution phase synthesis was reported at the 1999 IBC conference on peptide technologies, “Peptisyntha's method of producing GMP peptides on an Industrial scale”. The commercial scale solution phase involves a convergent synthesis, where the two fragments, Mpr-Har-Gly and Asp-Trp-Pro, are coupled to provide six of the seven residues needed for eptifibatide. The last residue attached is S-trityl-cysteinamide, as described, for example, in U.S. Pat. No. 5,506,362. After removal of the S-trityl protecting groups (on cysteinamide and mercaptopropionyl residues), and subsequent intramolecular cyclization via di-sulphide bond formation afforded crude eptifibatide with reported purity of about 80%. Two column purifications improve the purity to greater than 99%.
U.S. Pat. No. 7,674,768 (“the '768 patent”) discloses a (1+6) solution phase synthesis for the preparation of eptifibatide by first preparing the fragment of Har-Gly-Asp-Trp-Pro-Cys(NH2)—S—S-Mpr-OH and then coupling the Har with the Mpr, to yield crude eptifibatide with a purity of about 46%. The crude eptifibatide on primary preparative column purification with trifluoroacetic acid/acetonitrile-based system and then secondary preparative column purification with acetic acid/acetonitrile based system gives pure eptifibatide with a purity of 99%. The synthesis disclosed in the '768 patent is schematically represented as follows:

The '768 patent discloses multiple use of highly expensive chromatography technique such as preparative column chromatography to improve the purity of the resultant crude eptifibatide; which in turn result to an increase in the consumption of chromatography eluents and manufacturing cycle time, this leads to decrease in the product yield and manifold increase in production cost.
Patent publication WO2011079621 (“the '621 publication”) discloses a (3+4) solution phase synthesis for the preparation of eptifibatide by separate preparation of two protected fragments Mpr-Har-Gly-OH and Asp-Trp-Pro-Cys-NH2. The coupling of these two fragments provides protected linear peptide; the protected groups are removed and subsequent intramolecular ring closure by disulfide bond formation afforded crude eptifibatide with a purity of about 84%. The crude eptifibatide on preparative column purification with trifluoroacetic acid/acetonitrile-based system and then freeze drying gives the pure eptifibatide trifluoroacetate with a purity of 99.14% and contains about 0.17%, 0.29% and 0.22% of closely related impurities. The synthesis disclosed in the '621 publication is schematically represented as follows:
                The '621 publication has the following disadvantages, such as:            i) The product, eptifibatide trifluoroacetate salt obtained after preparative purification requires additional process steps such as another preparative purification to make pharmaceutically acceptable eptifibatide acetate salt,    ii) The benzyl group deprotection at -Asp is carried with hydrogen atmosphere in presence of palladium on carbon. The use of palladium for the debenzylation in sulphur containing compounds lead to insufficient conversion due to sulphur poisoning of the catalyst,    iii) It doesn't mention the content of isomeric impurities present/formed during the synthesis of the intermediates and the linear peptide, and    iv) The presence of closely related impurities is above the acceptable level recommended by the regulatory authorities.
U.S. Patent publication No 2006/0276626 (“the '626 publication”) discloses a process for the preparation of eptifibatide by first hexamer [Mpr-Har-Gly-Asp-Trp-Pro] was synthesized by solid phase synthesis using super acid labile resin and then introducing the Cys-NH2 by a solution phase synthesis, to yield crude eptifibatide. The crude eptifibatide on preparative column purification with trifluoroacetic acid system gives eptifibatide trifluoroacetate at a purity of 98.5% and then secondary preparative column purification for counter-ion exchange followed by lyophilization gives pure eptifibatide with a purity of 99%.
PCT publication WO 2009/150657 (“the '657 publication”) discloses a process for preparation of eptifibatide by using Fmoc solid phase synthesis. The '657 publication also discloses a purification process of crude eptifibatide by using preparative chromatography followed by salt exchange.
PCT publication WO 2004/092202 (“the '202 publication”) discloses a process for purification of eptifibatide by repeated purifications using preparative chromatography and then lyophilization.
It is known that purity and yield of the peptide are important aspects of any route of synthesis. Purity is represented by the degree of presence of pharmacologically active impurities, which though present in trace amounts only, may disturb or even render useless the beneficial action of the peptide when used as a therapeutic agent. The impurities can be unreacted starting materials, by-products of the reaction, products of side reactions, or racemization products.
Solution phase synthesis has generally been viewed as more feasible than solid phase synthesis for the large scale manufacture of eptifibatide. However, solubility issues, generation of racemization impurities and the formation of complex reaction mixtures present challenges for large scale solution phase synthesis. Complex reaction mixtures, for example, make purification of the product more difficult. Ways exist to overcome these problems, such as the use of persilylated amino acids and phase transfer reagents, as described, for example, in U.S. Pat. No. 4,954,616, and extensive chromatographic purification such as multiple preparative column chromatographies, as described in the known literature, are expensive and difficult to operate on an industrial scale.
None of the available literature teaches about the purity and impurity profile for the crude eptifibatide. In solution phase peptide synthesis repeated purification such as preparative chromatography is required to remove the impurities at crude stage; this involves consumption of large volumes of organic solvent as eluent, leads to a low yield of the final eptifibatide, which in turn result to an increase in the manufacturing cost. Thus, improvements in the synthesis such as lowering the inbuilt process impurities, leading to a robust process with minimal racemization from the initial stages of synthesis are needed.
Accordingly, there remains a need for an alternative processes to prepare eptifibatide to overcome the aforementioned difficulties, in a convenient and cost efficient manner and on a commercial scale.
The present invention provides alternate processes for the preparation of eptifibatide substantially free of racemization impurities using improved process modifications to minimize the racemization during the synthesis, as well as purification techniques that includes the use of either single preparative chromatography purification process with acetic acid as eluent or a simple flash chromatography as a substitute to expensive multiple preparative chromatography. The process of the present invention can be practiced on an industrial scale and also can be carried out without sacrifice of overall yield and purity of the product.